QuantiNova SYBR Green RT-PCR Kit

Internal control for positive in-process verification of successful RT-PCR

Accurate quantification over several logs of template

Ultrafast, universal protocol for all standard and fast cyclers

The QuantiNova SYBR Green RT-PCR Kit enables sensitive quantification of RNA targets by real-time one-step PCR using SYBR Green I detection. A combination of various integrated safety features removes variables and prevents artifacts, ensuring reliable gene expression profiling. The combination of a unique two-phase hot-start and PCR buffer system in the ready-to-use master mix allows room-temperature setup and ensures highly sensitive qRT-PCR on any real-time cycler. The QuantiNova Internal Control RNA can be used to test successful reverse transcription and amplification. It is intended to report instrument or chemistry failures, errors in assay setup and the presence of inhibitors. What's more, the visual indicator reduces pipetting error when performing ultrafast RT-PCR in less than one hour.

At ambient temperature the HotStaRTScript is inhibited by the RT-Blocker and the QuantiNova DNA Polymerase is kept inactive by QuantiNova Antibody and QuantiNova Guard. At 50°C the RT is activated while the QuantiNova DNA polymerase remains inactive. At 95°C the RT enzyme is denatured and the DNA polymerase is activated.

Accurate reaction setup indicated by the built-in pipetting control.

The master mix contains an inert blue dye. Combined with QuantiNova Yellow Template Dilution Buffer, the resulting solution turns green, indicating that the reaction was set up correctly.

Performance

The QuantiNova SYBR Green RT-PCR Kit uses a two-phase hot-start procedure (see figure “Principle of the novel QuantiNova two-phase hot-start mechanism”). This includes heat-mediated activation of both the HotStaRT-Script reverse transcriptase at 50°C and the PCR polymerase at 95°C. At low temperatures the HotStaRT-Script forms a complex with an RT-Blocker molecule, leading to inactivation. At 50°C, the complex dissociates and the active HotStaRT-Script enzyme is released. The QuantiNova DNA Polymerase is kept in an inactive state by the QuantiNova Antibody and a novel additive, QuantiNova Guard, which stabilizes the complex. This improves the stringency of the hot-start procedure and prevents extension of non-specifically annealed primers and primer–dimers. Within 2 minutes of raising the temperature to 95°C, the QuantiNova Antibody and QuantiNova Guard are denatured and the QuantiNova DNA Polymerase is activated, enabling PCR amplification. This unique two-phase hot-start enables reaction setup to remain stable for at least 2 hours at room temperature, preventing the creation of artifacts and also facilitating automated reaction setup.

The newly developed internal control is a defined transcript (RNA molecule) that can simply be added to your samples and transcribed into cDNA. It is intended to report instrument or chemistry failures, errors in assay setup or the presence of inhibitors. Since the internal control behaves comparably to real transcripts, it can be used to confirm successful reverse transcription and amplification.

The master mix supplied with the QuantiNova SYBR Green RT-PCR Kit contains an inert blue dye that does not interfere with the reverse transcription or real-time PCR, but increases visibility in the tube or well. The QuantiNova Yellow Template Dilution Buffer contains an inert yellow dye. When the template nucleic acid, diluted with the QuantiNova Yellow Template Dilution Buffer, is added to the master mix, the color of the solution changes from blue to green, providing a visual indication of correct pipetting and reaction setup (see figure “Accurate reaction setup indicated by the built-in pipetting control”).

The QuantiNova DNA Polymerase and the unique composition of the RT-PCR buffer provide sensitive quantification of low-copy RNA targets, as well as accurate quantification over a wide linear range on any common cycler.

Principle

The QuantiNova SYBR Green RT-PCR Kit contains an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of RNA targets using fluorescent dye SYBR Green I.
The kit comes with a unique PCR buffer that contains a balanced combination of K+ and NH4+ ions, which promotes specific primer annealing, enabling high specificity and sensitivity. In addition, the HotStaRT-Script enables cDNA synthesis from a wide range of RNA template amounts, while the QuantiNova DNA Polymerase provides a stringent hot-start procedure, preventing the formation of nonspecific products. The unique two-phase hot-start procedure allows reaction setup to remain stable for up to two hours at room temperature supporting automated reaction setup.

The built-in visual control allows you to check whether template has been added to the reaction and therefore prevents pipetting errors during reaction setup.

Detecting variations in cDNA synthesis allows you to check the reproducibility of your results. The newly developed internal control is a defined transcript (RNA molecule) that can be optionally added to your samples and transcribed into cDNA. It is intended to report instrument or chemistry failures, errors in assay setup and the presence of inhibitors.

Procedure

The QuantiNova SYBR Green RT-PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time consuming. Simply add primers, RNA template and RT-Mix to the ready-to-use SYBR Green RT-PCR master mix and start the reaction. Follow the protocol in the QuantiNova SYBR Green RT-PCR Kit Handbook to get fast and reliable results on any real-time cycler.

Applications

The QuantiNova SYBR Green RT-PCR Kit can be used for gene expression analysis of RNA targets on any real-time cycler. This includes the Rotor-Gene Q and instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche and Agilent.